Iron-sulfur [Fe-S] proteins are ubiquitous occurring in all life forms from the most primitive bacteria and archaea to the most advanced eucaryotes. These proteins are not only central to almost every essential biological electron transfer process but they also function in hydration/dehydration reactions, the generation and stabilization of radical intermediates, iron storage, oxidative stress and iron sensing and the regulation of gene expression. The general goal of the work proposed here is to elucidate the relationships between protein structure and [Fe- S] cluster structure, function, redox properties and reactivity. The experiments described are designed to address three fundamental questions in [Fe-S] protein metallobiochemistry. How does the structure of the protein control the reduction potential of a [4FE-4S]/2+/+ cluster? What is the mechanism of proton transfer from the solvent to a buried [3Fe-4S]0 cluster? What protein factors determine whether a protein will assemble a 3 Fe or a 4 Fe cluster and their interconversion? To answer these questions we have developed Azotobacter vinelandii ferredoxin I (FdI) as an [Fe-S] model system. This is an in depth, multi-disciplinary study where spectroscopic, direct electrochemical and computational methods are applied to [Fe-S] protein variants produced by site-directed mutagenesis, where the data are interpreted based on x-ray structures of mutant proteins and where the physiological consequences are studied by expressing proteins in their native background. The results are applicable to the entire class of [Fe-S] proteins and are of fundamental importance to efforts aimed at changing the reactivity of an existing protein or in the design of new [Fe-S] proteins. In addition to using it as a model we are also characterizing the function of AvFdI. This protein is greater than 90% identical to the 7Fe ferredoxins synthesize by a variety of Pseudomonas species including the important human pathogen P. aeruginosa. Our recent studies provide compelling evidence that FdI has a regulatory function related to an oxidative stress system that responds to the superoxide propagater paraquat. We have shown that FdI controls the expression of its redox partner NADPH:ferredoxin reductase indirectly through a regulatory cascade. Experiments described herein are designed to elucidate the mechanism of this regulation by identifying, purifying and characterizing the components involved in the oxidative stress regulatory system and by studying interactions of these components with each other and with possible effectors (e.g. superoxide, NO).